The cloning and expression of heterologous genes in fungi has been used to produce a variety of useful proteins. For example: Lambowitz, U.S. Pat. No. 4,486,533, discloses the autonomous replication of DNA vectors for filamentous fungi by mitochondrial plasmid DNA and the introduction and expression of heterologous genes into Neurospora; Yelton et al., U.S. Pat. No. 4,816,405, discloses tools and systems that enable the modification of important strains of filamentous ascomycetes to produce and secrete large quantities of desired heterologous proteins; Buxton et al., U.S. Pat. No. 4,885,249, discloses the transformation of Aspergillus niger by a DNA vector that contains a selectable marker capable of being incorporated into the host A. niger cells; and McKnight et al., U.S. Pat. No. 4,935,349, discloses a method for expressing higher eukaryotic genes in Aspergillus involving promoters capable of directing the expression of a heterologous gene in Aspergillus and other filamentous fungi. Similar techniques have been used to clone the mtr gene involved with amino acid transport in Neurospora crassa ("N. crassa") and to verify the tight linking of the cloned DNA to genomic markers flanking this gene in vivo. Stuart, W. D. et al., Genome (1988) 30:198-203; Koo, K. and Stuart, W. D. Genome (1991) 34:644-651.
Filamentous fungi possess many characteristics which make them good candidates for use in producing eukaryotic proteins. Filamentous fungi can secrete complex proteins; correctly fold three dimensional proteins including disulfide bond formation; proteolytically clip proteins following translation; and glycosylate proteins using n-linked and o-linked glycosylation reactions. These abilities have made this group of organisms attractive hosts for the production of secreted recombinant proteins. (MacKenzie, D. A. et al., J Gen Microbiol (1993) 139:2295-2307; Peberdy, J. F., Trends in BioTechnology (1994) 12:50-57).
Neurospora crassa has been used as a host cell for recombinant production of homologous and heterologous proteins such as those from mammals (see Yamashita, R. A. et al., Fungal Genetics Newsletter (1995 Suppl.) 42A for porcine relaxin; Kato, E. et al., Fungal Genetics Newsletter (1995 Suppl.) 42A for mammalian thrombolytic protein (mTh); Buczynski, S. et al. Fungal Genetics Newsletter (1995 Suppl.) 42A for human antibody light and heavy chains; and Nakano, E. T. et al. Fungal Genetics Newsletter (1993) 40:54-56 for bovine preprochymosin), plants (see Rasmussen-Wilson, S. J. et al., Appl. Environ. Microbiol. (1997) 63:3488-3493 for zeamatin from corn); fungi (see Carattoli, A., et al., Proc Nat Acad Sci USA (1995) 92:6612-6616 for neutral amino acid permease gene mtr expression; Puetz, D. Neurospora Newsletter (1984) 31:17 for ornithine transcarbamylase (OTS); Caru, M. et al. J. Appl. Bacteriol. (1989) 67:401-410 for invertase; Connerton, I. F. et al. Molec. Microbiol. (1990) 4:451-460 for acetyl-CoA synthetase with introns correctly spliced out; Arnaise, S. et al. Proc Nat Acad Sci USA (1993) 90:6616-6620 for mating-type genes; Hiett, K. L. et al. Mol. Gen. Genet. (1990) 222:201-205 for catabolic dehydroquinase, QUT E gene; and Weiss, R. L. et al. Gene Manipulations in Fungi, Academic Press Inc.:New York, pp. 280-292 (1985) for a review of expressing A. nidulans gene products), bacteria (see Patel, B. et al., J. Cell. Biochem. (1985) Suppl. 9C:172 (Abstr.) for the E. coli Ch1M nitrate gene), and mammalian viruses (see Sachs, M. S. et al., Nucleic Acids Res. (1997) 25:2389-2395 for herpes virus thymidine kinase).
Moreover, such recombinant protein production has occurred in Neurospora crassa via expression from heterologous promoters, such as that from Aspergillus (see Weiss et al., Supra), Podospora anserina (see Amaise et al., Supra), and a mammalian viral promoter (see Sachs et al., Supra), all of which suggests that Neurospora crassa can utilize regulatory elements of heterologous genes.
Additionally, Neurospora crassa has recently been used as a host cell for expressing recombinant heterodimeric and multimeric proteins by means of a heterokaryon., PCT Application WO 95/21263. A "heterokaryon" (or a heterokaryonic cell) is a cell formed from the fusion of two filamentous fungal parent strains, each heterokaryon cell thus containing two (or more) genetically different nuclei. Heterokaryons contain nuclei from two parent strains that are generally homozygous for all heterokaryon compatibility alleles (except for the mating type allele when the tol gene is present). At least ten chromosomal loci have been identified for heterokaryon incompatibility: het-c, het-d, het-e, het-i, het-5, het-6, het-7, het-8, het-9 and het-10, and more are inferred to exist. Perkins et al., "Chromosomal Loci of Neurospora crassa", Microbiological Reviews (1982) 46:426-570, at 478.
Aside from the expression of useful gene products, recombinant expression has been used to produce products resulting from a metabolic pathway, i.e. from the action of a number of gene products. For example, a diverse variety of polyketide synthases constitute various pathways for the production of the large family of polyketides, some of which have antibiotic or other pharmacological properties. Thus genes encoding the various polyketide synthases necessary for the production of particular polyketides, such as actinorhodin and aloesaponarin, have been recombinantly expressed in heterologous host cells (see Malpartida et al. Nature (1984) 309:462; Bartel et al. J. Bacteriol. (1990) 172:4816-4826). Moreover, host cells expressing hybrid polyketide synthases and hybrid polyketides have also been constructed (see Khosla et al. J. Bacteriol. (1993) 175:2197-2204; Hopwood et al. Nature (1985) 314:642-644; Sherman et al. J. Bacteriol. (1992) 174:6184-6190; U.S. Pat. No. 5,712,146).
Other metabolic pathways of interest in the art include those that produce primary and secondary metabolites as well as those that result in useful catabolic activities. Primary and secondary metabolites are respectively defined as those involved in the metabolic processes central to most cells and those involved in specialized cellular processes. Examples of primary metabolites are components involved in cellular biosynthetic machinery, energy production or utilization, and the turnover of cellular constituents, while secondary metabolites include substances such as antibiotics, anti-tumor or anti-cancer agents, anti-fungal agents, mating factors or pheromones, terpenes, toxins, alkaloids, biodegradable plastics, pigments, signaling molecules, cell surface molecules, secreted molecules, and numerous others. Useful catabolic activities include degradation or detoxification of waste materials, desulfurization of petroleum, and the breakdown of large or complex molecules such as cellulose or lignin.
The present invention advances the work of that disclosed in PCT Application WO 95/21263 by providing methods and compositions for producing a population of cells expressing heterologous gene products necessary for producing various metabolites and conducting various metabolic activities. Preferably, the cells are heterokaryonic filamentous fungi. Such methods and compositions are useful in the production of known products, such as proteins, nucleic acids and other metabolites, as well as the discovery and production of novel metabolites by the constitution of a novel combinatorial metabolic pathway in the cells.